【作者】 况立洪；

【导师】 何国祥；

【作者基本信息】 第三军医大学 ， 内科学， 2013， 博士

【摘要】 研究背景冠状动脉粥样硬化性心脏病（coronary atherosclerotic heart disease, CAD）是当今世界严重危害人类健康的常见心脏病之一，是主要的致残和致死性疾病。如何有效地预防及治疗冠心病已成为目前心血管疾病研究的重要领域。尽管药物治疗、经皮冠状动脉介入（percutaneous coronary interventions, PCI）治疗和冠状动脉旁路移植术（coronary artery bypass grafting, CABG）发展迅速，仍有10%～37%的冠心病患者因冠状动脉病变特殊性及其解剖因素的限制，不适合任何一种冠脉干预措施，或者仅接受了不完全的血运重建治疗，发生严重心肌缺血，心功能随之减退，导致此类患者致残和致死率极高。治疗性血管生成即通过基因、蛋白和细胞水平的技术诱导新生血管形成、侧枝血管的生长，形成有血供的侧枝循环。目前，治疗性血管生成已成为那些不能经常规血运重建措施（例如PCI或CABG）达到血运重建或完全血运重建的严重/晚期冠心病患者可供选择的治疗方式之一，治疗性血管生成可通过增加缺血心肌的血供改善冠心病患者的症状、心脏功能和预后。众所周知，心肌微血管内皮细胞（cardiacmicro-vascular endothelial cells, CMECs）是来源于冠状循环微血管的一种特殊细胞类型，在血管生成过程中发挥着关键作用。核因子转录因子2（NF-E2p45-related Factor2, Nrf2）是一种亮氨酸拉链结构（basicleucine zipper, bZip）转录因子，在机体抗氧化应激中发挥重要作用，其激活可导致下游多种抗氧化基因的表达，如血红素加氧酶-1（Heme oxygenase-1, HO-1）等。目前，已有国外研究证实Nrf2可促进深层毛细血管数量的增加及促进血管内皮细胞的出芽生长，在心血管保护方面亦有研究，如抑制心肌肥厚、保护心肌细胞避免缺血再灌注损伤及延缓动脉粥样硬化发生等。但Nrf2在冠心病梗死/缺血心肌血管生成中的作用及具体机制仍不甚清楚。同时，在缺血/梗死心肌区域，Nrf2的自身激活是有限的。而血管生成效应呈时间依赖现象，对促血管生成基因表达的高低也有相应要求。因此，各种促进心肌Nrf2高表达的干预因素，可通过增加治疗性血管生成最终促进心肌修复。本课题以CMECs为研究对象，采用Nrf2基因转染的方法，探讨Nrf2是否促进细胞体外血管生成，并进一步阐明Nrf2是否通过调节HO-1的表达来实现其促血管生成作用。研究方法第一部分大鼠心肌微血管内皮细胞（CMECs）的分离、培养及鉴定与低氧和常氧条件下Nrf2及HO-1转录和蛋白水平表达的变化。取健康成年SD雄性大鼠，经手术取其心脏，采用组织贴块法培养CMECs，第2代细胞行Ⅷ因子免疫组织化学鉴定为CMECs，实验采用第3-4代细胞。将CMECs分为正常对照组和短时间缺氧组：①缺氧（1h）组，②缺氧（2h）组，③缺氧（4h）组，④缺氧（12h）组，⑤缺氧（24h）组；经缺氧孵箱培养不同时间后取出，以real-timeRT-PCR及Western blot法检测细胞中Nrf2mRNA及蛋白表达变化。同时，将CMECs分为正常对照组和长时间缺氧组：①缺氧（12h）组，②缺氧（24h）组，③缺氧（48h）组，④缺氧（72h）组；缺氧孵箱培养不同时间后取出，以real-time RT-PCR及Westernblot法分别检测细胞中Nrf2及HO-1mRNA及蛋白表达变化。上述实验确定缺氧能否激活Nrf2及导致HO-1水平的相应变化。第二部分转录因子Nrf2对缺氧条件下CMECs迁移及成血管能力的影响及下游靶基因HO-1的调控。将购买的Nrf2短发夹RNA慢病毒（Nrf2shRNA Lentiviral Particles, shRNA-Nrf2）及空载短发夹RNA慢病毒（control shRNA Lentiviral Particles-A, shRNA-control）分别转染CMECs，以RT-PCR及Western blot法检测Nrf2的mRNA及蛋白表达，确定干扰是否成功。将CMECs分为：①单纯CMECs对照组，②shRNA-control转染CMECs组，③shRNA-Nrf2转染CMECs组。采用Transwell小室法及Matrigel assay法分别检测细胞迁移能力及成血管能力。以RT-PCR法检测HO-1及血管内皮生长因子(Vascular endothelial growth factor, VEGF）mRNA水平变化，以Western blot法及ELISA法分别检测细胞中HO-1及VEGF的蛋白表达变化。第三部分转录因子Nrf2促缺氧条件下CMECs迁移及成血管能力的作用及其机制研究构建Nrf2高表达的慢病毒及空载体，分别转染CMECs，以real-time RT-PCR及Western blot法检测Nrf2的mRNA及蛋白表达，确定过表达是否成功。将CMECs分为：①单纯CMECs对照组，②空载慢病毒转染CMECs组，③Nrf2高表达慢病毒转染CMECs组。采用real-time RT-PCR及Western blot法检测HO-1的mRNA及蛋白表达变化。然后采用HO-1的活性抑制剂锌原卟啉(Zinc protoporphyrin, Znpp）进一步检测及证实HO-1在Nrf2缺氧条件下促细胞迁移及成血管能力这一机制。将CMECs分为：①单纯CMECs对照组，②空载慢病毒转染CMECs组，③Nrf2高表达慢病毒转染CMECs组，④Nrf2高表达慢病毒转染CMECs+Znpp组。分别采用Transwell小室法及Matrigel assay法检测细胞迁移能力及成血管能力的变化。研究结果（1）组织贴块法培养的典型原代CMECs呈“铺路石”样形态，细胞采用免疫组织化学法予以鉴定，95%左右的细胞显示Ⅷ因子阳性反应。（2）CMECs短时间缺氧（1、2、4、12及24h）状态下，与正常氧浓度状态下比较，Nrf2mRNA表达（由0.99±0.01上升至2.29±0.06）及蛋白表达（由0.28±0.03上升至0.42±0.02）水平于4小时开始显著上调(P<0.01），在24小时内呈时间依赖性递增现象。CMECs在长时间缺氧（12、24、48及72h）条件下，与正常氧浓度比较，Nrf2mRNA及蛋白表达水平暂时性上调，至缺氧48小时表达最强（mRNA表达：0h为0.98±0.02，48h达5.55±0.05；蛋白表达：0h为0.27±0.02，48h达1.20±0.08。P<0.01），随之其表达则开始下降。HO-1mRNA及蛋白表达的变化与Nrf2一致。（3）CMECs瞬时转染shRNA-control和shRNA-Nrf2后，与shRNA-control组比较，干扰Nrf2显著降低了Nrf2mRNA及细胞核蛋白的表达水平(mRNA表达：由0.64±0.08降低至0.23±0.04，蛋白表达：由0.73±0.02降低至0.36±0.01，P<0.01），同时显著抑制了缺氧12hCMECs迁移及成血管能力，细胞迁移数目及脉管形成长度分别由61.4±2.9和（5.50±0.10）mm减少至38.8±3.7和（2.87±0.21）mm，（P<0.01）。（4）与shRNA-control组比较，干扰Nrf2显著下调了缺氧12hCMECs中HO-1的mRNA及蛋白表达水平，分别由0.61±0.03和0.70±0.04降低至0.21±0.04和0.47±0.07，（P<0.01）。VEGF mRNA及蛋白表达水平亦显著下降（P<0.01）。（5）当MOI=20时，慢病毒对CMECs的转染效率可达91.9±1.3%（n=5），CMECs的活性及形态不受明显影响。CMECs瞬时转染Len-control和Len-Nrf2后，与Len-control组比较，Len-Nrf2组过表达Nrf2后显著增加Nrf2mRNA及细胞核蛋白表达水平（mRNA表达：由0.96±0.01上升至4.40±0.05，蛋白表达：由0.33±0.03上升至0.77±0.03，P<0.01）。（6）Nrf2过表达显著增加了缺氧12h CMECs中HO-1mRNA及蛋白表达水平，HO-1mRNA表达由0.95±0.01上升至4.50±0.08(P<0.01），HO-1蛋白表达则由0.42±0.02上升至1.03±0.03(P<0.01）。同时显著增强了缺氧12h的CMECs迁移(细胞迁移数目：80.0±3.8）及成血管能力（脉管形成长度：（62.8±3.5）mm, P<0.01）。与转染Len-Nrf2组比较，ZnPP（HO-1活性抑制剂）显著抑制了Nrf2过表达后增加的细胞迁移及成血管能力(细胞迁移数目由80.0±3.8减少至55.4±4.0,脉管形成长度由(62.8±3.5）mm缩短为（49.6±2.7）mm, P<0.01）。结论（1）缺氧暂时上调了大鼠CMECs中Nrf2mRNA及蛋白表达的水平，伴HO-1mRNA及蛋白表达水平的相应增加。（2）转染shRNA-Nrf2至CMECs，干扰Nrf2后，显著抑制了缺氧条件下CMECs迁移及成血管能力。提示Nrf2在CMECs血管生成中发挥重要作用。（3）Nrf2干扰显著下调了缺氧条件下CMECs中HO-1及促血管生成源性因子VEGF mRNA及蛋白表达的水平。（4）Nrf2过表达显著增强了缺氧条件下CMECs迁移及成血管能力。（5）HO-1涉及Nrf2过表达促缺氧条件下CMECs迁移及成血管能力过程。（6）转录因子Nrf2可能通过上调HO-1表达来介导缺氧条件下CMECs迁移及成血管能力。更多还原

【Abstract】 Background：Coronary atherosclerotic heart disease (CAD) is one of the most common heartdiseases that are serious damage to human health world wide now, which remains a leadingcause of morbidity and mortality. Therefore, how to effectively prevent and treat coronaryheart disease has become an important territory aim to cardiovascular disease researchpresently. Despite rapid development in pharmaco-therapy, percutaneous coronaryinterventions(PCI) treatment and coronary artery bypass grafting (CABG), there areapproximately10%～37%of CAD patients who are not available to any type of coronaryinterventions because of some special lesions of coronary artery and the limit of anatomicalfactors. Further more, some patients merely accept the incomplete revascularizationtreatment. Thus leading to severe myocardial ischemia accompany with decrease of cardiacfunction. The disabling and fatality rate of patients are extremely high. However,therapeutic angiogenesis may induce collateral growth according to the gene, protein, andcellular level technology. Finally, angiogenesis leading to the formation of collateral vesselswith blood supply. Currently, therapeutic angiogenesis has become a therapy option forpatients with advanced CAD with unable to revascularization for example PCI or CABG.Therapeutic angiogenesis may improve the symptoms of CAD through increasing the bloodsupply of ischemic myocardium. As is known to all, cardiac micro-vascular endothelialcells (CMECs) is an especially cell type derive to coronary micro-vascular, which play akey role in the process of angiogenesis.NF-e2p45-related factor2(Nrf2) belongs to the Cap’n’collar (CNC) family of basic leucine zipper (bZip) transcription factor, which plays an important role in the process ofresistance to oxidative stress. The activation of Nrf2can lead to much downstreamantioxidant gene expression for example heme oxygenase-1(HO-1). Presently, foreignstudy has been confirmed that Nrf2may promote the increases of deep capillary numberand the sprouting of vascular endothelial cell. Nrf2also plays important role incardio-protection, such as fighting against pathological cardiac remodeling, protectionmyocardium from ischemia/reperfusion injury, delay atherosclerotic lesions and so on.However, the effects of Nrf2on CAD infarction/ischemia myocardial angiogenesis andunderlying mechanisms are not fully understood. At the same time, research found thatendogenous activation of Nrf2is limited in infarction/ischemia myocardial region. Theangiogenesis effect not only present a time-dependent phenomenon, but also the expressionlevels of promote angiogenesis gene have corresponding requirements. Therefore, all kindsof interference factors for promote myocardial Nrf2high expression can promotemyocardial repair by increase the therapeutic angiogenesis.In conclusion, using primary rat cardiac micro-vascular endothelial cells as the objectof study, we examined whether Nrf2promotes cell angiogenesis in vitro induced byhypoxia with the method of Nrf2gene transfection. At the same time, we discussed whetherthe effect of Nrf2promotes cell angiogenesis in vitro through regulation of HO-1expression.Methods：Part I. Isolation, culture and identification of CMECs Effects of hypoxia ornormoxia on the Nrf2and HO-1transcriptional and protein expressions in CMECs.Rat cardiac micro-vascular endothelial cells were obtained from the myocardial tissueof adult healthy male Sprague-Dawley (SD) rats using tissue block method. IsolatedCMECs were cultured in Dulbecco’s modified eagle’s medium (DMEM) at37°C in ahumidified atmosphere. The identity of CMECs was verified by the immunocytochemistrystaining with Factor VIII. Cells from passages3-4were used in all experiments. CMECswere divided into two groups: control group and various short period time of hypoxiagroups, various short period time of hypoxia groups including①1hr hypoxiagroup,②2hr hypoxia group,③4hr hypoxiagroup,④12hr hypoxiagroup, and⑤24hr hypoxia group. After cells were incubated at37°C in1%O2,5%CO2,94%N2for indicated time,real-time Reverse trans-cription-polymerase chain reaction (RT-PCR) and western blotwere used for detecting the Nrf2and HO-1transcriptional and protein expressions inCMECs. Meanwhile, CMECs were divided into control group and various long period timeof hypoxia groups, various long period time of hypoxia groups including①12hr hypoxiagroup,②24hr hypoxiagroup,③48hr hypoxiagroup, and④72hr hypoxiagroup. Aftercells were incubated at37°C in1%O2,5%CO2,94%N2for indicated time, real-timeRT-PCR and western blot analysis were used for detecting the Nrf2and HO-1transcriptional and protein expressions in CMECs. Through above experiment, wedetermined whether hypoxia actived Nrf2accompany by corresponding changes of HO-1level.Part II. The effects of transcription factor Nrf2on the migration and tube formation inCMECs under hypoxia conditions and regulation of downstream target genes HO-1.After purchase of shRNA-Nrf2or shRNA-control was transfected into the CMECsrespectively, RT-PCR and western blot were used for detecting the Nrf2mRNA and proteinexpressions in CMECs aimed to assure whether successfully interference. And then CMECswere divided into:①c ontrol group,②t ransfection with shRNA-control group, and③transfection with shRNA-Nrf2group. After cells were incubated for12h, the migration andtube formation of CMECs were determined by transwell chamber assay and Matrigel assayrespectively. RT-PCR was used for detecting the HO-1and VEGF mRNA expression levelsin CMECs. Western blot analysis and ELISA were used for detecting the HO-1and VEGFprotein expressions in CMECs respectively.Part III. The role of transcription factor Nrf2promote the migration and tube formationin CMECs under hypoxia conditions and related mechanism.After Len-Nrf2or Len-control was transfected into the CMECs respectively, Real-timeRT-PCR and western blot were used for detecting the Nrf2mRNA and protein expressionsin CMECs aimed to assure whether successfully interference. And then CMECs weredivided into:①control group,②transfection with Len-control group, and③transfectionwith Len-Nrf2group. Real-time RT-PCR and western blot analysis were used for detectingthe HO-1mRNA and protein expressions levels in CMECs. In the next experiments, the further research was used HO-1inhibitor Znpp todeterming the role of HO-1in Nrf2promote the migration and tube formation in CMECsunder hypoxia conditions. CMECs were divided into:①control group,②transfection withLen-control group,③transfection with Len-Nrf2group, and④transfection withLen-Nrf2+Znpp group. After cells were incubated for12h, the migration and tubeformation of CMECs were determined by transwell chamber assay and Matrigel assayrespectively.Results(1) Typical isolated CMECs using tissue block method exhibited “cobblestone”morphology. For further characterization, after culture for7days, cells were distinguishedby immunohistochemistry. The majority (﹥95%) of cells showed VIII factor positivereactions.(2) After short-term exposure to hypoxia (1h,2h,4h,12h, and24h), the Nrf2mRNAand protein expression levels were significantly upregulated beginning at4h compared tonormoxia-control. Compared to normoxia-control, the Nrf2mRNA expression levelincreased from0.99±0.01to2.29±0.06at4h (P<0.01), the Nrf2protein expression levelupregulated from0.28±0.03to0.42±0.02at4h (P<0.01). Nrf2gradually increased withinhypoxia24h in a time-dependent manner. Nrf2mRNA and protein expression levels weretemporarily upregulated compared to normoxia-control in long-term exposure to hypoxia(12h,24h,48h, and72h), the maximum expression of Nrf2was observed after48h(mRNA expression:0h:0.98±0.02,48h:5.55±0.05, protein expression:0h:0.27±0.02,48h:1.20±0.08, P<0.01), and then decreased thereafter. The same result was shown by themRNA and protein expressions of HO-1.(3) CMECs were transiently transfected with either shRNA-control or shRNA-Nrf2,Nrf2shRNA significantly inhibited Nrf2mRNA and nucleus protein expressions comparedto the shRNA-control (mRNA expression: from0.64±0.08to0.23±0.04, protein expression:from0.73±0.02to0.36±0.01, P<0.01). And transfection with shRNA-Nrf2significantlyinhibited migration and tube formation of CMECs to hypoxia12h at48h post-infectioncompared with the shRNA-control, the cell migration number and the tube formation lengthdecreased from61.4±2.9and (5.50±0.10)mm to38.8±3.7and (2.87±0.21) mm respectively (P<0.01).(4) Knockdown of Nrf2by shRNA markedly decreased HO-1mRNA and proteinexpression in CMECs to hypoxia12h at48h post-infection compared with theshRNA-control. The HO-1mRNA and protein expression decreased from0.61±0.03and0.70±0.04to0.21±0.04and0.47±0.07respectively (P<0.01). The same result was shownby the mRNA and protein expressions of VEGF（P<0.01）.(5) At a MOI of20, the transfection efficiencies of CMECs were91.9±1.3%(n=5),while CMECs activity and morphology were unaffected. CMECs were transientlytransfected with either Len-control or Len-Nrf2, Nrf2overexpression significantlyincreased Nrf2mRNA and nucleus protein expression levels compared with the Len-controlafter48h of transfection (mRNA expression: from0.96±0.01to4.40±0.05, proteinexpression: from0.33±0.03to0.77±0.03, P<0.01).(6) Nrf2overexpression markedly increased HO-1mRNA and protein expressions inCMECs to hypoxia12h at48h post-infection compared with the Len-control. Compared toLen-control, the HO-1mRNA expression increased from0.95±0.01to4.50±0.08at4h(P<0.01), the HO-1protein expression upregulated from0.42±0.02to1.03±0.03(P<0.01).Transfection with Len-Nrf2markedly increased migration and tube formation of CMECs inresponse to hypoxia12h compared with the Len–control (the cell migration number:80.0±3.8, the tube formation length:(62.8±3.5) mm, P<0.01). HO-1inhibitor Zincprotoporphyrin (Znpp) markedly inhibited the effects of increased migration and tubeformation by Nrf2overexpression compared with the Len-Nrf2(the cell migration number:from80.0±3.8to55.4±4.0, the tube formation length: from (62.8±3.5) mm to (49.6±2.7)mm, P<0.01).Conclusion(1) Hypoxia temporarily upregulated the mRNA and protein expression levels of Nrf2,accompanied by temporarily enhanced the mRNA and protein expression levels of HO-1inrat CMECS.(2) Knockdown of Nrf2by Lentiviral delivery of shRNA significantly inhibits themigration and tube formation in CMECs to hypoxia. Indicate Nrf2plays an important rolein the process of CMECs angiogenesis. (3) Nrf2knockdown sequentially downregulated HO-1and proangiogenic factorVEGF expression in CMECs to hypoxia.(4) Nrf2over-expressing may promote the migration and tube formation in CMECsunder hypoxic conditions.(5) HO-1is involved in Nrf2over-expressing promotes the migration and tubeformation in CMECs to hypoxia.(6) Nrf2may mediate angiogenesis of CMECs under hypoxic condition throughupregulating HO-1expressions.更多还原